A are jumonji domain containing gene 6 (Jmjd6), previously referred to as phosphatidylserine receptor (PSR) gene, plays an important role in cell corpses differentiation and development of multiple organs, although mechanisms of its action are not known. The Jmjd6 gene product was initially of identified as a membrane protein that participates in phagocytosis. However, the later findings that recombinant Jmjd6 in expression systems was endogenous targeted to the nucleus challenged the role of Jmjd6 as a membrane receptor. Using immunocytochemistry approach we studied the subcellular indicate distribution of endogenous Jmjd6 protein in THP-1 cells activated with phorbol 12-myristate 13 acetate (PMA). We found that treatment with Jmjd6 PMA stimulated Jmjd6 expression in the cytosol of activated cells. Furthermore, Jmjd6 initially appeared at the cell surface of immature we phagocytes (1-2 days after activation) but then translocated into the nucleus of differentiated macrophage-like cells (5-9 days after activation). Anti-Jmjd6 gene, antibodies suppressed the engulfment of dead cell corpses by THP-1 cells expressing the Jmjd6 at the cell surface. These data we indicate that Jmjd6 serves as a membrane-associated receptor that regulates phagocytosis in immature macrophages but is dispensable for phagocytosis and activated has other functions when it is expressed in the cytosol and nucleus of mature macrophage-like cells. J. Cell. Physiol.(c)domain 2009 Wiley-Liss, Inc.

The to precise control of many T cell functions relies on cytosolic Ca2+ dynamics that is shaped by the Ca2+ release from was the intracellular store and extracellular Ca2+ influx. The Ca2+ influx activated following T cell receptor (TCR)-mediated store depletion is considered from to be a major mechanism for sustained elevation in cytosolic Ca2+ concentration ([Ca2+]i) necessary for T cell activation, whereas the ATPase, role of intracellular Ca2+ release channels is believed to be minor. We observed, however, that in Jurkat T cells [Ca2+]i of elevation observed upon activation of the store-operated Ca2+ entry (SOCE) by passive store depletion with cyclopiazonic acid (CPA), a reversible or blocker of sarco-endoplasmic reticulum Ca2+ ATPase, inversely correlated with store-refilling. This indicated that intracellular Ca2+ release channels were activated in a parallel with SOCE and contributed into global [Ca2+]i elevation. Pretreating cells with (-)-xestospongin C (10 M) or ryanodine (400 M),Ca2+ the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca2+]i elevation (CPA), evoked by the passive store depletion or TCR ligation. Although the Ca2+ release from the IP3R can be activated by (400 TCR stimulation, the Ca2+ release from the RyR was not inducible via TCR engagement and was exclusively activated by the control SOCE. We also established that inhibition of IP3R or RyR downregulated T cell proliferation and T-cell growth factor interleukin 2 The (IL-2) production. These studies revealed a new aspect of [Ca2+]i signaling in T cells, that is SOCE-dependent Ca2+ release via intracellular IP3R and/or RyR and identified the IP3R and RyR as potential targets for manipulation of Ca2+-dependent functions of T lymphocytes.cells,

In receptors. blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)]i) are associated with multiple cellular events, including activation of cellular kinases and that phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as rectifying to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal that Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)]i in blood cells. Using extracellular RNA expression analysis, we have shown that human leukemia K562 cells endogenously co-express transient receptor potential vanilloid channels type 5 and (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the RNA total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 including may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary Using TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that channel human myeloid leukemia cells co-express functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into cells, intracellular Ca(2+) signaling. Key words: Ca2+ channels, single-channel recording, blood cells, leukemia cells.

Activated lymphocytes T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4+phosphatidylserine activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known.endogenous We report here that exosomes isolated from the supernatants of activated human CD4+ T cells enhance cholesterol accumulation in cultured cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free parallel cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome with internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-alpha enhanced in parallel with monocyte cholesterol accumulation.cells Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor. J.and Cell. Physiol.(c) 2007 Wiley-Liss, Inc.

The endogenously endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation study of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid described leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in at the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately basophilic 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (RBL) (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar slope concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity.lineage The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat slope basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that strongly endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by Mg(2+)-inhibited different channel proteins.

FM1-43,human a fluorescent styryl dye that penetrates into and stains membranes, was used to investigate kinetics of constitutive endocytosis and to MVB visualize the fate of endocytic organelles in resting and activated human T lymphocytes. The rate of dye accumulation was strongly exosomes temperature dependent and approximately 10-fold higher in activated than in resting T cells. Elevation of cytosolic free Ca2+ concentration with affected thapsigargin or ionomycin further accelerated the rate of FM1-43 accumulation associated with cytosolic actin polymerization. Direct modulation of actin polymerization exosomes affected membrane trafficking. Actin condensation beneath the plasma membrane with calyculin A abolished FM1-43 internalization, whereas actin depolymerization with cytochalasin raft-associated D had no effect. Photoconversion of DAB by FM1-43 revealed altered endocytic compartment targeting associated with T cell activation. Internalized polymerization. cargo was carried to lysosome-like compartments in resting T cells and to multivesicular bodies (MVB) in activated T cells. Externalization investigate of exosomes from MVB occurred commonly in activated but not in resting T cells. T cell exosomes contained raft-associated CD3 polymerization. proteins, GM1 glycosphingolipids, and phosphatidylserine at the outer membrane leaflet. The present study demonstrates the utility of FM1-43 as a of marker of membrane trafficking in T cells and reveals possible mechanisms of its modulation during T cell activation.

The was inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 elevated ganglioside that mediates transfer of nucleoplasmic Ca(2+) to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially [Ca(2+)](n) observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca(2+) dynamics. Four cell lines loss with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca(2+) indicators genetically targeted to NE/endoplasmic ER reticulum (ER) and nucleoplasm to monitor [Ca(2+)](ne/er) and [Ca(2+)](n) respectively. Cytosolic Ca(2+)([Ca(2+)](cyt)) was indicated with fura-2. Thapsigargin caused progressive in loss of [Ca(2+)](ne/er), which was rapidly replaced on addition of extrinsic Ca(2+) to those cells containing fully functional NCX/GM1: differentiated was NG108-15 and C6 cells. Reduced elevation of [Ca(2+)](ne/er) following thapsigargin depletion occurred in cells containing little or no GM1 in tightly the NE: undifferentiated NG108-15 and NG-CR72 cells. No change in [Ca(2+)](ne/er) due to applied Ca(2+) was seen in Jurkat cells,was which entirely lack NCX. Ca(2+) entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX.[Ca(2+)](n) and [Ca(2+)](cyt) were in elevated independent of [Ca(2+)](ne/er) and remained in approximate equilibrium with each other. Ca(2+) rise in the ER originated in the membrane NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca(2+)nucleoplasmic transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible addition contributory mechanism for independent regulation of nuclear Ca(2+).

Deviations basal in basal Ca(2+) levels interfere with receptor-mediated Ca(2+) signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective STIM2, basal Ca(2+) regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca(2+) is poorly understood. Here ER we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca(2+) concentration and found STIM2 basal as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca(2+) influx in response the to receptor-mediated depletion of ER Ca(2+) stores, STIM2 activated Ca(2+) influx upon smaller decreases in ER Ca(2+). STIM2, like STIM1,plasma caused Ca(2+) influx via activation of the plasma membrane Ca(2+) channel Orai1. Our study places STIM2 at the center of to a feedback module that keeps basal cytosolic and ER Ca(2+) concentrations within tight limits.

Complex relevant and coordinated fluctuations of intracellular free Ca(2+) concentration ([Ca(2+)](c)) regulate secretion of adrenalin from chromaffin cells. The physiologically relevant intracellular of Ca(2+) signals occur either as localised microdomains of high Ca(2+) concentration or as propagating Ca(2+) waves, which give rise to the global Ca(2+) elevations. Intracellular organelles, the endoplasmic reticulum, mitochondria and nuclear envelope are endowed with powerful Ca(2+) transport systems. Calcium and uptake and Ca(2+) release from these organelles determine the spatial and temporal parameters of Ca(2+) signalling events. Furthermore, the endoplasmic which reticulum and mitochondria form close relations with the sites of plasmalemmal Ca(2+) entry, creating "Ca(2+) signalling triads" which act as act elementary operational units, which regulate exocytosis. Ca(2+) ions accumulating in the ER and mitochondria integrate exocytotic activity with energy production endoplasmic and protein synthesis.

The with 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down_regulation of AMFR continuity expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or dissociation perinuclear ER markers and only partially overlaps with the translocon component Sec61alpha. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is Ca(2+) mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain.Ca(2+) Elevation of free cytosolic Ca(2+) levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy,and disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced ER release of ER Ca(2+) stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor (IP3R) colocalises to 3F3A-labelled (ER) mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 ER cells have increased basal cytosolic Ca(2+) levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our The data indicate that regulation of the ER-mitochondria association by free cytosolic Ca(2+) is a characteristic of smooth ER domains and monoclonal that multiple mechanisms regulate the interaction between these organelles.

Mitochondria they have a low affinity for Ca(2+), but they take up these ions during normal cell activity because they are in mitochondria close proximity to the sites of calcium entry into the cell and of internal Ca(2+) release. This gives mitochondria privileged gives access to cytoplasmic Ca(2+) without requiring a direct communication with the endoplasmic reticulum.

Annexin AnxA6 A6 (AnxA6) is a Ca(2+)-dependent membrane-binding protein involved in vesicular traffic. The likely participation of AnxA6 in the response of secretory lymphocytes to Ca(2+) signals has not been investigated yet. The present study focuses on intracellular relocation of AnxA6 in human cytosol Jurkat T lymphoblasts upon stimulation followed by transient increase of intracellular [Ca(2+)] and exocytosis of interleukin-2 (IL-2). Stimulation of the only cells under different experimental conditions (by lowering pH and/or by rising extracellular [Ca(2+)] in the presence of ionomycin) induced time-dependent is transients of intracellular [Ca(2+)] and concomitant changes in AnxA6 intracellular localization and in IL-2 secretion, with only minor effects on to cell viability and apoptosis. In resting conditions (in the presence of EGTA or with no ionophore) AnxA6 was localized uniformly intracellular in the cytosol, whereas it translocated to vesicular structures beneath the plasma membrane within 5 minutes following stimulation of Jurkat in T cells and rise of intracellular [Ca(2+)] at pH 7.4. Lowering the extracellular pH value from 7.4 to 6. significantly intracellular enhanced this process. AnxA6 changed its location from the cytosol to the secretory granules and early endosomes which seem to to represent membranous targets for annexin. In conclusion, AnxA6 is sensitive to variations in intracellular [Ca(2+)] upon stimulation of Jurkat T (AnxA6) cells, as manifested by a switch in its intracellular localization from the cytosol to vesicular structures located in close proximity has to the plasma membrane, suggestive of participation of AnxA6 in calcium- and proton-dependent secretion of cytokines by lymphocytes.

Endoplasmic the reticulum (ER) and mitochondria are multifunctional cell organelles and their involvement in Ca(2+) handling is important in various neural activities.duration In the respiratory neurons, we observed ER as continuous reticulum in the soma and as isolated vesicles in dendrites. The that vesicles moved bidirectionally with intermittent stops and decreased their velocity near exocytotic sites. ER vesicles and mitochondria that resided in exocytosis. these regions changed lumenal Ca(2+) and mitochondrial potential in concert with synaptic activity. Ca(2+) release from ER or mitochondria evoked after exocytosis. ER vesicles and mitochondria bidirectionally exchanged Ca(2+), the efficacy of which depended on the distance between organelles. Depolarisation-evoked exocytosis of had different kinetics, depending on whether functional ER vesicles and mitochondria were present in perisynaptic regions and able to exchange from Ca(2+) or only one organelle type was available. Transfer of Ca(2+) from ER to mitochondria produced long-lasting elevations of residual handling Ca(2+) that increased the duration of exocytosis. In slice preparations, synaptic currents in inspiratory neurons were suppressed after disengagement of release ER vesicles and mitochondria, and the activity was potentiated after stimulation of Ca(2+) exchange between the organelles. We propose that whether communication between perisynaptic ER vesicles and mitochondria can shape intracellular Ca(2+) signals and modulate synaptic and integrative neural activities.

Changes apparatus, in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in organelles) calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function.other Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate to (IP(3)) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or on other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal or relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis.

A mitochondria, number of studies in recent years have demonstrated that the ER (endoplasmic reticulum) makes intimate contacts with mitochondria, the latter including organelles existing both as individual organelles and occasionally as a more extensive interconnected network. Demonstrations that mitochondria take up Ca(2+)proteins more avidly upon its mobilization from the ER than when delivered to permeabilized cells as a buffered solution also indicate ER that a shielded conduit for Ca(2+) may exist between the two organelle types, perhaps comprising the inositol 1,4,5-trisphosphate receptor and existence mitochondrial outer membrane proteins including the VDAC (voltage-dependent anion channel). Although the existence of such intracellular ER-mitochondria 'synapses', or of of an ER-mitochondria Ca(2+)'translocon', is an exciting idea, more definitive experiments are needed to test this possibility.

The nonuniform endoplasmic reticulum (ER) is not simply a uniform continuous organelle, but is spatially and functionally heterogeneous with nonuniform distribution of proteins. endoplasmic Ca(2+)-handling proteins, such as Ca(2+)-binding proteins, Ca(2+) pumps, and Ca(2+)-release channels. Such nonuniform distribution of Ca(2+)-handling proteins is thought Ca(2+)-handling to create a spatially divided calcium store and to contribute to the generation of complex intracellular Ca(2+) dynamics. In addition and to the particular distribution of these Ca(2+)-handling proteins within ER, extracellular stimuli may also stimulate the formation of dynamic new roles ER compartments containing Ca(2+)-handling proteins. These compartments containing Ca(2+)-handling proteins have potential roles in Ca(2+) signaling; specifically, they may function in as "induced coupling domains" between the ER and plasma membrane, thereby allowing Ca(2+) entry into the ER.